This project aims to assess the importance of changes in intracellular free calcium concentration, [Ca++]i, and intracellular pH, pHi, as key regulators in the early stages of the growth and proliferation of lymphocytes and certain cultured cell lines. The importance of both these ion levels has been widely hypothesized, but largely on inconclusive indications such as the dependence of cell growth on extracellular Ca++ or pH, or the influence of Ca ionophores, or the effect of Ca++ or pH on cell extracts. Until very recently, [Ca++]i could not be measured at all in small cells capable of proliferation, and pHi could not be measured with good time resolution and noninterference from internal organelles. However, recent techniques developed by this investigator have now permitted rapid, continuous, quantitative readout of [Ca++]i and pHi in intact lymphocytes and other mammalian cell types. The keys to the methodology are (1) new, fluorescent, membrane-impermeant indicators highly specific and sensitive either to [Ca++]i or pHi near their normal intracellular resting values, and (2) the invention of ester derivatives which are membrane-permeant and which hydrolyze intracellularly, trapping the indicators in the cytoplasm of intact cells. These techniques have produced the first demonstration that several T-cell mitogens cause early increases in [Ca++]i in mouse thymocytes and pig node lymphocytes. Moreover, all agents found to oppose mitogen-induced rises in [Ca++]i are known to inhibit mitogenesis, with the intriguing exception of the tumor promoter, tetradecanoylphorbol acetate. This project would extend the preliminary results by comparing [Ca++]i in different lymphocyte subpopulations, examining a wider range of mitogenic stimuli, inhibitors, and lymphokines, quantitatively comparing [Ca++]i with early metabolic events and later proliferation, and exploring the mechanism of action of phorbol esters. Tecnical goals include the further improvement of Ca++-indicators and the measurement of [Ca++]i in single cells. Measurements of [Ca++]i and pHi would also be made in other proliferating systems such as cultured cells transformed by temperature-sensitive tumor viruses, or permanent tumor cell lines. Such investigations would add to our understanding of epigenetic controls of cell proliferation, both normal and pathological.